FEATURED NEW PRODUCTS

Copper-Free Protein Detection in Live Cells — Click-iT® DIBO Reagents

what they are
The use of copper in labeling and detection reactions can be harmful to cells, reduce the fluorescence of certain fluorophores, and impair the activity of some enzymes. Our new Click-iT® DIBO reagents now offer a means of achieving click reactions without copper, enabling live-cell surface labeling of proteins and sugars using click chemistry and gentle conjugation of azide-labeled material.

what they offer

Preservation of protein function and cell viability

Efficiency—reaction is complete in 1 hour

Stability—reaction product contains an irreversible, covalent bond

how they work
Click chemistry employs a highly specific bioorthogonal reactive chemistry for the in situ labeling of biomolecules. The classic click reaction involves copper-catalyzed triazole formation from an azide and an alkyne, but our new DIBO reagents permit the labeling of azide-modified macromolecules without the metal catalysts, which enables live-cell detection and prevents protein damage. Nine new products are available for copper-free detection, including DIBO derivatives of Alexa Fluor® dyes, TAMRA dye, and biotin labels, and reactive probes capable of modifying amine, cysteine, and carboxy groups.

The azide and DIBO moieties are interchangeable; the molecule can be labeled with a DIBO and reacted with a fluorophore or hapten-azide.

C10405,C10406,C10407,C10408,C10410,C10411,C10412,C10413,C10414

Light Up Mitochondria — CellLight® Mitochondria Reagents

what they are
CellLight® reagents are fluorescent protein–signal peptide fusions that permit accurate and specific targeting to cellular structures, including mitochondria, for live-cell imaging or fixed-cell analyses. It’s now easier than ever to visualize mitochondria in combination with other probes, such as the rhod calcium indicators.

what they offer

Efficient transduction of mammalian cells, including "difficult" cells such as primary and stem cells

Multiplex analysis with cellular stains, including other CellLight® reagents, antibodies, or MitoTracker® and LysoTracker® dyes

Simplicity—add, incubate, and image

how they work
Cellular labeling with CellLight® reagents employs BacMam technology, which uses a modified insect cell baculovirus coupled with a mammalian promoter as a vehicle to efficiently deliver and express genes in mammalian cells. Unlike expression vectors, BacMam reagents enable titratable and reproducible expression and offer high cotransduction efficiency, enabling multiple BacMam reagents to be used in the same cell.

HeLa cells were transduced with CellLight® Mitochondria-GFP (green). The following day, cells were loaded with 5 µM X-Rhod-1 AM (red) for 30 min at room temperature. Cells were imaged using standard FITC/TRITC filters on a DeltaVision® Core microscope. The red fluorescence of the high-affinity calcium indicator X Rhod-1 (Kd of 700 nM) is well resolved from green-fluorescent probes and is good for detecting small changes in cytosolic calcium levels.

what it is
SAA is an acute-phase protein that may be significantly increased following inflammation, infection, or injury. The SAA Mouse ELISA kit is specific for quantifying SAA protein in mouse serum or plasma samples, while the SAA Multispecies ELISA Kit can be used for bovine, porcine, canine, feline, or equine samples.

what it offers

Fast results—only one wash step, and incubation is only 1.25 hours

Specificity—sandwich ELISA using 2 antibodies against SAA for greater specificity

Ease of use—recommended sample dilutions are provided

how it works
This convenient kit format saves you time by providing a simple protocol and a precoated 96-well plate. Add your diluted serum or plasma sample, standards, and controls to the 96-well plate and incubate for 1 hour. After incubation, perform a single wash step, incubate for 15 minutes with substrate, add stop solution, and your plate is ready to read.

NEW APPLICATIONS

Reveal Elusive Nucleosome Kinetics Using Click Chemistry

A recent publication in Science (Science (2010) 328:1161) describes a new technique, “CATCH-IT”, that enables researchers to label and capture sites of histone replacement, which in turn can reveal genome-wide kinetics of nucleosomes.

Briefly, cells are fed the methionine analog L-azidohomoalanine (Click-iT® AHA), which incorporates into proteins during active protein synthesis. Click-iT® AHA contains a very small modification: an azido moiety, which enables detection of newly synthesized proteins through a “click chemistry” reaction—formation of a copper-catalyzed triazole from the azide and a biotin alkyne. Following the click reaction and isolation of nuclei, histone proteins containing the biotin tag are affinity-purified using streptavidin-based magnetic beads. The universal histone replacement variant H3.3 is then used to measure nucleosome turnover.

Click chemistry describes an extremely powerful class of reactions that occur between biologically unique moieties (e.g., an azide and an alkyne). Click reactions have several benefits: the reaction between the detection moieties is efficient; no extreme temperatures or solvents are required; the reaction product is stable; and the components of the reaction are bioinert, which means that no side reactions occur. This final point is the greatest advantage of this powerful technique; click chemistry–labeled molecules can be applied to complex biological samples and detected with high sensitivity.

DEPARTMENTS

Buzzworthy

Autophagy is the recycling of cytosolic and organelle components during times of starvation, a cellular function that is critical to survival. The process begins with the formation of the autophagosome—a multilamellar body that has captured a volume of cytosol. This structure then fuses with a lysosome to degrade the contents, which are then transported back into the cytosol for use by the cell. To clarify conflicting reports regarding the origins of the autophagosome, Hailey and colleagues observed its formation using a series of organelle-specific, fusion protein–expressing constructs and organelle-staining probes (LysoTracker® DND99 and MitoTracker® Red CMXRos). The team was able to determine that starvation-induced autophagosomes emerge from mitochondrial outer membranes. They further concluded that starvation-induced autophagosomes are distinct from those formed in response to ER stress or calcium perturbation, and that they do not arise as a result of mitophagy.

Increases in intracellular calcium (Ca2+) control a diverse set of cellular processes. Even within a single cell, the magnitude and duration of a change in the level of Ca2+ can vary greatly. Observing these concentration changes within a cell requires a diverse portfolio of detection reagents. We offer a complete range of reagents for calcium imaging applications.

When choosing a dye for a particular imaging application, dye emission intensities are a key consideration. The green-fluorescent fluo family of dyes has a very low emission at rest, resulting in a large increase in emission intensity upon Ca2+ binding, but making it difficult to resolve the cell. Members of the Oregon Green® BAPTA dye family have a higher resting fluorescence and therefore can be used to locate cells and structures; however, the maximal change in emission intensity is lower upon Ca2+ binding.

Generally, upon low-intensity stimulation, cells recruit a small number of plasma membrane or intracellular release channels. High-affinity dyes such as fluo-4, fluo-3, Oregon Green® BAPTA-1, and Oregon Green® BAPTA-2 are best-suited for these kinds of applications. A response to medium-intensity stimulation of cells, where many plasma membrane or intracellular channels are recruited for prolonged periods of time, is best detected using medium-affinity dyes such as fluo-5F. For high-intensity stimulation, low-affinity dyes such as Oregon Green® BAPTA-5N and fluo-4FF are most useful.

Choose red Ca2+ dyes for imaging cells or tissues with high autofluorescence, for simultaneous imaging of a green fluorophore, or for studies of calcium signaling, where these dyes enable simultaneous imaging of Ca2+ and a GFP chimera. As with green-emitting dyes, we offer both high-affinity red-emitting dyes (rhod-3 and X-rhod-1) and low-affinity dyes such as X-rhod-5F.

Imaging Ca2+ signaling in HeLa cells. HeLa cells were loaded with 5 µM rhod-3 AM (red), followed by 5 µM mag-fluo-4 (green), both at room temperature. Cells were then stimulated with histamine (100 µM) to release Ca2+ from the ER (decrease in mag-fluo-4 trace, green) into the cytoplasm (increase in rhod-3 signal, red).

Research Tools for Influenza Confirmation and Analysis
Life Technologies supplies the tools, reagents, and support necessary for whole-genome sequencing, resequencing, and antiviral resistance testing of influenza viruses. For antiviral resistance testing, the NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit includes everything you need to quantitate neuraminidase activity and neuraminidase inhibitor resistance of avian, equine, human, and porcine influenza viruses. Check back for new kits available in September!